This proposal investigates how the neural retina regulates the development of polarity and barrier function in the retinal pigment epithelium (RPE). It tests the hypothesis that during development, retinal-derived factors, induce the maturation of tight junctions. This maturation is required for the RPE to form the outer blood-retinal barrier. Reestablishing this barrier is the goal of therapies aimed at repairing RPE damaged by surgical intervention or disease. Barrier function requires the RPE to develop two related, but distinct properties. First, it must form tight junctions, a barrier to diffusion between the choroid and the subretinal space. Second, it must polarize the distribution of proteins that regulate transport across the barrier. In most epithelia, these properties are induced by interactions at the basal and lateral membranes. In RPE, this induction is modulated by interaction with the neural retina at the apical membrane. Novel interactions between the neural retina and the RPE were revealed by examining the gradual development of polarity during embryogenesis. Furthermore, these studies revealed a previously undescribed maturation of the tight junctions. The current proposal will reconstitute this developmental process in culture. RPE will be isolated from chick embryos before the outer blood-retinal barrier forms. These immature cells will be cultured with medium that was conditioned by retinas isolated from older embryos, when the barrier is forming. The retinal-derived factors will be characterized according to functional properties: the ability to increase barrier function, and the ability to induce the changes in junction structure and composition that occur in vivo. Accordingly, the study will provide the first molecular and morphological description of tight junctions as they are converted from a leaky to a tight form in vivo. The maturation of tight junctions will be monitored with electron microscopic and immunofluorescence techniques to examine changes in morphology, and with immunofluorescence and immunobiochemical techniques to examine the assembly of the tight junction proteins 7H6, occludin, cingulin and ZO-l. The functional assays will be use to characterize and purify the retinal factors on an analytical scale. These studies address fundamental issues of epithelial cell biology, and examine an aspect of RPE/retinal interactions that have been previously ignored. This information is required to understand the function of RPE in normal tissue and in proliferative disease. These studies may help identify factors that promote the regeneration of a functional monolayer from endogenous or transplanted RPE.